Bacillus thuringiensis crystal polypeptides, polynucleotides, and compositions thereof

ABSTRACT

The present invention provides insecticidal polypeptides related to  Bacillus  Cry2 polypeptides. Nucleic acids encoding the polypeptides of the invention are also provided. Methods for using the polypeptides and nucleic acids of the invention to enhance resistance of plants to insect predation are encompassed.

This application is a divisional of, and claims benefit to, U.S.application Ser. No. 11/675,729, filed Feb. 16, 2007 now U.S. Pat. No.7,737,117, which application is a divisional of, and claims benefit toU.S. application Ser. No. 11/067,557, filed, Feb. 25, 2005 now U.S. Pat.No. 7,208,474, which claims priority to and benefit of U.S. provisionalapplication No. 60/547,664, filed Feb. 25, 2004, all of which areincorporated herein by reference in their entirety.

1. FIELD OF THE INVENTION

The present invention relates generally to the field of pest control andprovides insecticidal polypeptides related to Bacillus Cry2 polypeptidesand the polynucleotides that encode them. The present invention alsorelates to methods and compositions for altering resistance of plants toinsect predation including, but not limited to, transgenic plantproduction.

2. BACKGROUND OF THE INVENTION

Numerous commercially valuable plants, including common agriculturalcrops, are susceptible to attack by insect and nematode pests. Thesepests can cause substantial reductions in crop yield and quality.Traditionally, farmers have relied heavily on chemical pesticides tocombat pest damage. However, the use of chemical pesticides raises itsown set of problems, including the cost and inconvenience of applyingthe pesticides. Furthermore, chemical residues raise environmental andhealth concerns. For these and other reasons there is a demand foralternative insecticidal agents.

An environmentally friendly approach to controlling pests is the use ofpesticidal crystal proteins derived from the soil bacterium Bacillusthuringiensis (“Bt”), commonly referred to as “Cry proteins.” Many ofthese proteins are quite toxic to specific target insects, but harmlessto plants and other non-targeted organisms. Some Cry proteins have beenrecombinantly expressed in crop plants to provide pest-resistanttransgenic plants. Among those, Bt-transgenic cotton and corn have beenwidely cultivated.

A large number of Cry proteins have been isolated, characterized andclassified based on amino acid sequence homology (Crickmore et al.,1998, Microbiol. Mol. Biol. Rev., 62: 807-813). This classificationscheme provides a systematic mechanism for naming and categorizing newlydiscovered Cry proteins.

It has generally been found that individual Cry proteins possessrelatively narrow activity spectra with the exception of Cry2A. Cry2A isunusual in that this subset of Cry proteins possesses a broadereffective range that includes toxicity to both the Lepidoptera andDiptera orders of insects. The Cry2A protein was discovered to be atoxin showing a dual activity against Trichoplusia ni (cabbage looper)and Aedes taeniorhynchus (mosquito) (Yamamoto and McLaughlin, 1982,Biochem. Biophys. Res. Comm. 130: 414-421). The nucleic acid moleculeencoding the Cry2A protein (termed Cry2Aa) was cloned and expressed inB. megaterium and found to be active against both Lepidoptera andDiptera insects (Donovan et al. 1988, J. Bacteriol. 170: 4732-4738). Anadditional coding sequence homologous to Cry2Aa was cloned (termedCry2Ab) and was found to be active only against Lepidoptera larvae(Widner and Whiteley, 1989, J Bacteriol 171:2).

Second generation transgenic crops could be more resistant to insects ifthey are able to express multiple and/or novel Bt genes. Accordingly,new insecticidal proteins having broad activity spectra would be highlydesirable.

3. SUMMARY OF THE INVENTION

The present invention relates to a novel Cry2 polypeptide, Cry2Ax (SEQID NO:2), isolated from Bacillus thuringiensis. Also encompassed by thepresent invention are Cry2Ax-derived polypeptides (SEQ ID NOS:4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224,226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,254, 256, 258, 260). In addition to the polypeptide sequence of Cry2Axand Cry2Ax-derived polypeptides, it will be appreciated thatpolypeptides of the invention also encompass variants thereof,including, but not limited to, any fragment, analog, homolog, naturallyoccurring allele, or mutant thereof. Polypeptides of the invention alsoencompass those polypeptides that are encoded by any Cry2Ax orCry2Ax-derived nucleic acid of the invention. In one embodiment,polypeptides that have at least one Cry2Ax functional activity (e.g.,insecticidal activity) and are at least 85%, 90%, 95%, 97%, 98%, or 99%identical to the polypeptide sequence of any of SEQ ID NOS:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224,226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,254, 256, 258, 260, or variants thereof. In another embodiment,polypeptides are encompassed that have at least one Cry2Ax functionalactivity (e.g., insecticidal activity), are at least 25, 50, 75, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,475, 500, 525, 550, 575, 600, or 625 contiguous amino acids in length,and are encoded by a polynucleotide that hybridizes under stringentconditions to the nucleic acid that encodes any of SEQ ID NOS: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222,224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250,252, 254, 256, 258, 260, or a variant thereof. Methods of production ofthe polypeptides of the invention, e.g., by recombinant means, are alsoprovided. Compositions comprising one or more polypeptides of theinvention are also encompassed.

The present invention also relates to the nucleic acid molecules ofCry2Ax (SEQ ID NO:1) and Cry2Ax-derived nucleic acid molecules (SEQ IDNOS:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191,193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219,221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,249, 251, 253, 255, 257, 259). Also encompassed by the present inventionare fragments and analogs which encode polypeptides that are at leastpartially functionally active, i.e., they are capable of displaying oneor more known functional activities associated with a wild type Cry2Axpolypeptide. In one embodiment, the invention encompasses an isolatednucleic acid molecule that comprises a nucleotide sequence i) which isat least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to thenucleotide sequence of any of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231,233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259;ii) that hybridizes with a nucleic acid probe consisting of thenucleotide sequence of any of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231,233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, ora complement thereof under stringent conditions; and/or iii) thatcomprises a nucleic acid molecule that encodes a polypeptide comprisingthe amino acid sequence of any of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230,232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258,260. Vectors comprising nucleic acids of the invention are alsoencompassed. Cells or plants comprising the vectors of the invention arealso encompassed.

The present invention also relates to transgenic plants expressing anucleic acid and/or polypeptide of the invention. The transgenic plantscan express the transgene in any way known in the art including, but notlimited to, constitutive expression, developmentally regulatedexpression, tissue specific expression, etc. Seed obtained from atransgenic plant of the invention is also encompassed.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows insecticidal activity of DNA clones from the second roundof shuffling. Each clone was expressed in N. benthamiana leaves usingforced infiltration. Each leaf disk was fed to a single 3^(rd) instar H.zea larvae. Following a 24-hour incubation period, the feeding activitywas determined by visual observation and expressed as an approximatefraction of leaf area remaining. The y-axis is the percent of the leafdisk remaining after exposure to the insect. The x-axis is the cloneexpressed in the leaf disk. Several clones shoed increased insecticidalactivity such as 7K (D_S01000779) (SEQ ID NO:10), 15K (D_S00999080) (SEQID NO:12), 16K (D_S01000269) (SEQ ID NO:14), 16R (D_S01037143) (SEQ IDNO:16), and 473R (D_S01037677) (SEQ ID NO:18).

FIG. 2 shows insecticidal activity of first round shuffled clone 44(D_S00503970) and third round shuffled clone D_S01764701. Each clone wasexpressed in N. benthamiana leaves using forced infiltration. Each leafdisk was fed to a single 3^(rd) instar H. zea larva. Following a 24-hourincubation period, the feeding activity was determined by video captureof the leaf disk. The y-axis is the number of pixels present in thecaptured leaf disk image. The x-axis is the clone expressed in the leafdisk. Results are shown for the average of three experiments. For eachexperiment at least eight leaf disks were tested for each clone.

FIGS. 3A-3B show efficacy results for transgenic tobacco plantsexpressing first round shuffled clone 44 in the plastid compartment(left panels) or in the cytoplasm (right panels). The efficacy of (A) H.zea or (B) S. exigua inhibition was determined after incubation of theleaves with the worms for 24 hours. The amount of leaf remaining wasobserved with video capture equipment for actual calculation of relativeleaf area remaining (number of pixels). Each transgenic plant had sixleaf disks taken for analysis. Because twenty five transgenic plantswere made using each transgene construct, the numbers distinguishdifferent plants using a particular construct.

FIGS. 4A-4B show transgene expression levels in the first round shuffledclone 44-expressing transgenic plants. This shuffled Cry2-derivedpolypeptide was expressed in (A) the plastidic or (B) cytoplasmicsubcellular compartments by transformation with pMAXY5469 or pMAXY5471,respectively. Western blot analysis was performed on transgenic plantextracts using a polyclonal antibody directed to the toxin region of thefirst round shuffled clone 44 polypeptide. Negative controls wereextracts taken from an untransformed plant. Positive controls wereeither 20 ng or 40 ng of purified Cry2Ax toxin. The molecular weight ofthe positive control Cry2Ax differs from that of the Cry2Ax-derivedpolypeptide in the plant extracts because the former is trypsinactivated and the latter is pro-toxin.

5. DETAILED DESCRIPTION

The present invention provides insecticidal polypeptides related toBacillus Cry2 polypeptides. Nucleic acid molecules encoding thepolypeptides of the invention are also provided. Methods for using thepolypeptides and nucleic acids of the invention to enhance resistance ofplants to insect predation are encompassed.

5.1 Polypeptides of the Invention

The present invention relates to a novel Cry2 polypeptide, Cry2Ax (SEQID NO:2), isolated from Bacillus thuringiensis. Also encompassed by thepresent invention are Cry2Ax-derived polypeptides (SEQ ID NOS: 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224,226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,254, 256, 258, 260). Polypeptides of the invention also encompass thosepolypeptides that are encoded by any Cry2Ax or Cry2Ax-derived nucleicacid of the invention (see Section 5.2).

In addition to the polypeptide sequence of Cry2Ax and Cry2Ax-derivedpolypeptides, it will be appreciated that polypeptides of the inventionalso encompass variants thereof, including, but not limited to, anysubstantially similar sequence, any fragment, analog, homolog, naturallyoccurring allele, or mutant thereof. Variants encompassed by theinvention are polypeptides that are at least partially functionallyactive, i.e., they are capable of displaying one or more knownfunctional activities associated with a wild type Cry2Ax polypeptide.Such functional activities include, but are not limited to, biologicalactivities, such as insecticidal activity; antigenicity, i.e., anability to bind or compete with Cry2Ax for binding to an anti-Cry2Axantibody; immunogenicity, i.e., an ability to generate antibody whichbinds to a Cry2Ax polypeptide. In some embodiments, the variants have atleast one functional activity that is substantially similar to itsparent polypeptide (e.g., a variant of Cry2Ax will have at least onefunctional activity that is substantially similar to Cry2Ax). As usedherein, the functional activity of the variant will be considered“substantially similar” to its parent polypeptide if it is within onestandard deviation of the parent.

In one embodiment, polypeptides that have at least one Cry2Ax functionalactivity (e.g., insecticidal activity) and are at least 85%, 90%, 95%,97%, 98%, or 99% identical to the polypeptide sequence of any of SEQ IDNOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162,164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190,192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218,220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246,248, 250, 252, 254, 256, 258, 260 are encompassed by the invention. Suchpolypeptides of the invention contain at least 1, at least 5, at least10, at least 20, at least 30, or all 40 amino acid residues from thegroup consisting of H₂, S₇, Q₂₇, Q₃₅, E₃₆, K₄₃, D₄₄, N₄₅, D₅₁, A₅₈, V₆₉,R₇₈, N₇₉, K₉₉, T₁₁₈, V₁₂₄, E₁₂₅, R₁₂₉, N₁₃₈, R₁₃₉, A₁₄₁, T₁₆₂, Q₁₆₅,M₁₆₆, L₁₈₃, I₁₉₂, H₂₁₁, R₂₁₃, R₂₁₇, D₂₁₈, V₃₂₄, E₃₈₆, T₃₉₉, S₄₀₅, Q₄₄₅,I₅₅₁, S₅₈₇, I₅₆₁, L₆₁₀, and L₆₃₁. The subscript indicates the amino acidresidue position corresponding to the position in SEQ ID NO:2 uponoptimal alignment of the polypeptide sequence with SEQ ID NO:2. Withrespect to an amino acid sequence that is optimally aligned with areference sequence, an amino acid “corresponds” to the position in thereference sequence with which the residue is paired in the alignment.

As used herein, where a sequence is defined as being “at least X %identical” to a reference sequence, e.g., “a polypeptide at least 95%identical to SEQ ID NO:2,” it is to be understood that “X % identical”refers to absolute percent identity, unless otherwise indicated. Theterm “absolute percent identity” refers to a percentage of sequenceidentity determined by scoring identical amino acids or nucleic acids asone and any substitution as zero, regardless of the similarity ofmismatched amino acids or nucleic acids. In a typical sequence alignmentthe “absolute percent identity” of two sequences is presented as apercentage of amino acid or nucleic acid “identities.” In cases where anoptimal alignment of two sequences requires the insertion of a gap inone or both of the sequences, an amino acid residue in one sequence thataligns with a gap in the other sequence is counted as a mismatch forpurposes of determining percent identity. Gaps can be internal orexternal, i.e., a truncation. Absolute percent identity can be readilydetermined using, for example, the Clustal W program, version 1.8, June1999, using default parameters (Thompson et al., 1994, Nucleic AcidsResearch 22: 4673-4680).

In another embodiment, fragments of Cry2Ax and Cry2Ax-derivedpolypeptides are encompassed by the invention. Polypeptides areencompassed that have at least one Cry2Ax functional activity (e.g.,insecticidal activity), are at least 25, 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525,550, 575, 600, or 625 contiguous amino acids in length of any of SEQ IDNOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162,164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190,192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218,220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246,248, 250, 252, 254, 256, 258, 260, and are encoded by a polynucleotidethat hybridizes under stringent conditions to the nucleic acid thatencodes any of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236,238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260. Inembodiments where the fragment of the invention encompasses any of theamino acid residues that correspond to amino acid residues 2, 7, 27, 35,36, 43, 44, 45, 51, 58, 69, 78, 79, 99, 118, 124, 125, 129, 138, 139,141, 161, 165, 166, 183, 192, 211, 213, 217, 218, 324, 386, 399, 405,445, 551, 587, 591, 610, 631 of SEQ ID NO:2, such polypeptides of theinvention contain at least 1, at least 5, at least 10, at least 20, atleast 30, or all 40 amino acid residues from the group consisting of H₂,S₇, Q₂₇, Q₃₅, E₃₆, K₄₃, D₄₄, N₄₅, D₅₁, A₅₈, V₆₉, R₇₈, N₇₉, K₉₉, T₁₁₈,V₁₂₄, E₁₂₅, R₁₂₉, N₁₃₈, R₁₃₉, A₁₄₁, T₁₆₂, Q₁₆₅, M₁₆₆, L₁₈₃, I₁₉₂, H₂₁₁,R₂₁₃, R₂₁₇, D₂₁₈, V₃₂₄, I₃₈₆, T₃₉₉, S₄₀₅, Q₄₄₅, I₅₅₁, S₅₈₇, I₅₉₁, L₆₁₀,and L₆₃₁.

In a specific embodiment, a fragment of the invention corresponds to thelength of the processed pro-toxin. There is a 5-6 kDa difference inmolecular weight between full length pro-toxin Cry2 and the processedCry2 toxin. This is the result of ˜40 amino acids being cleaved from thepro-toxin Cry2 polypeptide (Rukmini et al., 2000, Biochimie 82:109-116;Aronson et al., 1993, Mol. Microbiol. 7:489-496; Morse et al., 2001,Structure 9:409-17). Polypeptides that correspond to this processed Cry2fragment can be provided in the methods of the present inventiondirectly to circumvent the need for pro-toxin processing.

In another specific embodiment, a fragment of the invention correspondsto a Cry2 domain. Cry2 polypeptides have three domains including i)domain I which is involved in insertion into the insect apical midgutmembrane and affects ion channel function, ii) domain II which isinvolved in receptor binding on the insect midgut epithelial cellmembrane, and iii) domain III which is involved in ion channel function,receptor binding, and insertion into the membrane (Dean et al., 1996,Gene 179:111-117; Schnepf et al., 1998, Microbiol. Molec. Biol. Rev.62:775-806).

In another embodiment, analog polypeptides are encompassed by theinvention. Analog polypeptides may possess residues that have beenmodified, i.e., by the covalent attachment of any type of molecule tothe Cry2Ax or Cry2Ax-derived polypeptides. For example, but not by wayof limitation, an analog polypeptide of the invention may be modified,e.g., by glycosylation, acetylation, pegylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand or other protein,etc. An analog polypeptide of the invention may be modified by chemicalmodifications using techniques known to those of skill in the art,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Furthermore, ananalog of a polypeptide of the invention may contain one or morenon-classical amino acids.

Methods of production of the polypeptides of the invention, e.g., byrecombinant means, are also provided (see Section 5.6).

Compositions comprising one or more polypeptides of the invention arealso encompassed. The compositions of the invention can further compriseadditional agents including, but not limited to, spreader-stickeradjuvants, stabilizing agents, other insecticidal additives, diluents,agents that optimize the rheological properties or stability of thecomposition, such as, for example, surfactants, emulsifiers,dispersants, and/or polymers.

5.2 Nucleic Acids of the Invention

The present invention also relates to the nucleic acid molecules ofCry2Ax (SEQ ID NO:1) and Cry2Ax-derived nucleic acid molecules (SEQ IDNOS:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191,193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219,221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,249, 251, 253, 255, 257, 259). Nucleic acid molecules of the inventionalso encompass those nucleic acid molecules that encode any Cry2Ax orCry2Ax-derived polypeptide of the invention (see Section 5.1).

In addition to the nucleic acid molecule of Cry2Ax and Cry2Ax-derivednucleic acid molecules, it will be appreciated that nucleic acids of theinvention also encompass variants thereof, including, but not limited toany substantially similar sequence, any fragment, homolog, naturallyoccurring allele, or mutant thereof. Variant nucleic acid moleculesencompassed by the present invention encode polypeptides that are atleast partially functionally active, i.e., they are capable ofdisplaying one or more known functional activities associated with awild type Cry2Ax polypeptide. Such functional activities include, butare not limited to, biological activities, such as insecticidalactivity; antigenicity, i.e., an ability to bind or compete with Cry2Axfor binding to an anti-Cry2Ax antibody; immunogenicity, i.e., an abilityto generate antibody which binds to a Cry2Ax polypeptide. In someembodiments, the variants have at least one functional activity that issubstantially similar to its parent nucleic acid molecule (e.g., avariant of a Cry2Ax nucleic acid molecule will encode a polypeptide thathas at least one functional activity that is substantially similar tothe polypeptide encoded for by the Cry2Ax nucleic acids molecule). Asused herein, the functional activity of the variant will be considered“substantially similar” to its parent polypeptide if it is within onestandard deviation of the parent.

In one embodiment, nucleic acid molecules that are at least 70%, 75%,80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to any of the nucleicacid molecules of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237,239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259 are encompassed bythe invention. Such nucleic acid molecules of the invention encodepolypeptides that contain at least one, at least 5, at least 10, atleast 20, at least 30, or all 40 amino acid residues from the groupconsisting of H₂, S₇, Q₂₇, Q₃₅, E₃₆, K₄₃, D₄₄, N₄₅, D₅₁, A₅₈, V₆₉, R₇₈,N₇₉, K₉₉, T₁₁₈, V₁₂₄, E₁₂₅, S₁₂₉, N₁₃₈, S₁₃₉, A₁₄₁, T₁₆₂, Q₁₆₅, M₁₆₆,L₁₈₃, I₁₉₂, H₂₁₁, S₂₁₃, S₂₁₇, D₂₁₈, V₃₂₄, I₃₈₆, T₃₉₉, S₄₀₅, Q₄₄₅,I_(551, S) ₅₈₇, I₅₉₁, L₆₁₀, and L₆₃₁.

To determine the percent identity of two nucleic acid molecules, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in the sequence of a first nucleic acid molecule for optimalalignment with a second or nucleic acid molecule). The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical overlappingpositions/total number of positions×100%). In one embodiment, the twosequences are the same length.

The determination of percent identity between two sequences can also beaccomplished using a mathematical algorithm. A non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (Karlin and Altschul, 1990, Proc.Natl. Acad. Sci. 87:2264-2268, modified as in Karlin and Altschul, 1993,Proc. Natl. Acad. Sci. 90:5873-5877). Such an algorithm is incorporatedinto the NBLAST and XBLAST programs (Altschul et al., 1990, J. Mol.Biol. 215:403 and Altschul et al., 1997, Nucleic Acid Res.25:3389-3402). Software for performing BLAST analyses is publiclyavailable, e.g., through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always>0)and N (penalty score for mismatching residues; always<0). For amino acidsequences, a scoring matrix is used to calculate the cumulative score.Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=⁻4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989, PNAS,89:10915).

The Clustal V method of alignment can also be used to determine percentidentity (Higgins and Sharp, 1989, CABIOS. 5:151-153) and found in theMegalign program of the LASERGENE bioinformatics computing suite(DNASTAR Inc., Madison, Wis.). The “default parameters” are theparameters pre-set by the manufacturer of the program and for multiplealignments they correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10,while for pairwise alignments they are KTUPLE 1, GAP PENALTY=3, WINDOW=5and DIAGONALS SAVED=5. After alignment of the sequences, using theClustal V program, it is possible to obtain a “percent identity” byviewing the “sequence distances” table on the same program.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

In another embodiment, fragments of Cry2Ax and Cry2Ax-derived nucleicacid molecules are encompassed by the invention. Nucleic acid moleculesare encompassed that have at least one Cry2Ax functional activity (e.g.,insecticidal activity), are at least 100, 250, 500, 750, 1000, 1500, or1800 contiguous nucleotides in length of any of SEQ ID NOS: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,253, 255, 257, 259, and/or hybridize under stringent conditions to thenucleic acid molecule that encodes any of SEQ ID NOS:2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228,230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256,258, 260. In embodiments where the nucleic acid fragment of theinvention encodes a polypeptide that encompasses any of the amino acidresidues that correspond to amino acid residues 2, 7, 27, 35, 36, 43,44, 45, 51, 58, 69, 78, 79, 99, 118, 124, 125, 129, 138, 139, 141, 161,165, 166, 183, 192, 211, 213, 217, 218, 324, 386, 399, 405, 445, 551,587, 591, 610, 631 of SEQ ID NO:2, such nucleic acids molecules of theinvention contain coding sequences for at least 1, at least 5, at least10, at least 20, at least 30, or all 40 amino acid residues from thegroup consisting of H₂, S₇, Q₂₇, Q₃₅, E₃₆, K₄₃, D₄₄, N₄₅, D₅₁, A₅₈, V₆₉,R₇₈, N₇₉, K₉₉, T₁₁₈, V₁₂₄, E₁₂₅, S₁₂₉, N₁₃₈, S₁₃₉, A₁₄₁, T₁₆₂, Q₁₆₅,M₁₆₆, L₁₈₃, I₉₂, H₂₁₁, S_(213, S) ₂₁₇, D₂₁₈, V₃₂₄, I₃₈₆, T₃₉₉, S₄₀₅,Q₄₄₅, I₅₅₁, S₅₈₇, I₅₉₁, L₆₁₀, and L₆₃₁.

In a specific embodiment, a fragment of the invention corresponds to thelength of nucleic acid that encodes the processed pro-toxin. There is a5-6 kDa difference in molecular weight between full length pro-toxinCry2 and the processed Cry2 toxin. This is the result of ˜40 amino acidsbeing cleaved from the pro-toxin Cry2 polypeptide (Rukmini et al., 2000,Biochimie 82:109-116; Aronson et al., 1993, Mol. Microbiol. 7:489-496;Morse et al., 2001, Structure 9:409-17).

In another specific embodiment, a fragment of the invention encodes apolypeptide that corresponds to a Cry2 domain.

In another embodiment, a nucleic acid molecule that hybridizes understringent conditions to any one of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259 is encompassed by the invention. Such nucleic acid molecules of theinvention encode polypeptides that contain at least 1, at least 5, atleast 10, at least 20, at least 30, or all 40 amino acid residues fromthe group consisting of H₂, S₇, Q₂₇, Q₃₅, E₃₆, K₄₃, D₄₄, N₄₅, D₅₁, A₅₈,V₆₉, R₇₈, N₇₉, K₉₉, T₁₁₈, V₁₂₄, E₁₂₅, R₁₂₉, N₁₃₈, R₁₃₉, A₁₄₁, T₁₆₂,Q₁₆₅, M₁₆₆, L₁₈₃, I₁₉₂, H₂₁₁, R₂₁₃, R₂₁₇, D₂₁₈, V₃₂₄, I₃₈₆, T₃₉₉, S₄₀₅,Q₄₄₅, I₅₅₁, S₅₈₇, I₅₉₁, L₆₁₀, and L₆₃₁.

The phrase “stringent conditions” refers to hybridization conditionsunder which a nucleic acid will hybridize to its target nucleic acid,typically in a complex mixture of nucleic acid, but to essentially noother nucleic acids. Stringent conditions are sequence-dependent andwill be different in different circumstances. Longer nucleic acidshybridize specifically at higher temperatures. Extensive guides to thehybridization of nucleic acids can be found in the art (e.g., Tijssen,Techniques in Biochemistry and Molecular Biology—Hybridization withNucleic Probes, “Overview of principles of hybridization and thestrategy of nucleic acid assays” (1993)). Generally, highly stringentconditions are selected to be about 5-10° C. lower than the thermalmelting point (T_(m)) for the specific nucleic acid at a defined ionicstrength pH. Low stringency conditions are generally selected to beabout 15-30° C. below the T_(m). The T_(m) is the temperature (underdefined ionic strength, pH, and nucleic acid concentration) at which 50%of the probes complementary to the target hybridize to the targetnucleic acid at equilibrium (as the target nucleic acids are present inexcess, at T_(m), 50% of the probes are occupied at equilibrium).Hybridization conditions are typically those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, and preferably 10 times background hybridization. In oneembodiment, stringent conditions include at least one wash (usually 2)in 0.2×SSC at a temperature of at least about 50° C., usually about 55°C., or sometimes 60° C. or 65° C., for 20 minutes, or substantiallyequivalent conditions. In a specific embodiment, the nucleic acidmolecule of the invention specifically hybridizes following at least onewash in 0.2×SSC at 55° C. for 20 minutes to a polynucleotide encodingthe polypeptide of any of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206,208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234,236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260. Inanother embodiment, stringent conditions include hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C. followed by one ormore washes in 0.2×SSC, 0.1% SDS at 50-65° C.

The phrase “specifically hybridizes” refers to the binding, duplexing,or hybridizing of a molecule only to a particular nucleotide sequenceunder stringent hybridization conditions when that sequence is presentin a complex mixture (e.g., total cellular or library DNA or RNA).

Vectors comprising nucleic acids of the invention are also encompassed.Cells or plants comprising the vectors of the invention are alsoencompassed.

The term “nucleic acid” or “nucleic acid molecule” herein refer to asingle or double-stranded polymer of deoxyribonucleotide orribonucleotide bases read from the 5′ to the 3′ end. It includeschromosomal DNA, self-replicating plasmids and DNA or RNA that performsa primarily structural role. The term “encoding” refers to apolynucleotide sequence encoding one or more amino acids. The term doesnot require a start or stop codon. An amino acid sequence can be encodedin any one of six different reading frames provided by a polynucleotidesequence and its complement.

Table 1 discloses Cry2Ax and Cry2Ax-derived sequences and thecorresponding sequence identity number.

5.3 Cry2Ax-Derived Sequences

Cry2Ax-derived polypeptides and nucleic acids of the invention can becreated by introducing one or more nucleotide substitutions, additionsand/or deletions into the nucleotide sequence of Cry2Ax or relatednucleic acids, such that one or more amino acid substitutions, additionsand/or deletions are introduced into the encoded protein. Generally,Cry2Ax-derived sequences are created in order to accentuate a desirablecharacteristic or reduce an undesirable characteristic of a Cry2Axpolypeptide. In one embodiment, Cry2Ax-derived polypeptides haveimproved insecticidal activity over Cry2Ax including, but not limitedto, greater potency and/or increased insect pest range. In anotherembodiment, Cry2Ax-derived polypeptides are expressed better than Cry2Axincluding, but not limited to, increased half life, less susceptible todegradation, and/or more efficient transcription or translation.

In one embodiment, a Cry2Ax (SEQ ID NO:1) nucleic acid molecule is usedas a template to create Cry2Ax-derived nucleotides. In anotherembodiment, a Cry2Ax related nucleic acid is used as a template tocreate Cry2Ax-derived nucleotides. In a specific embodiment, Cry2Ab* isused as a template. Cry2Ab* has two amino acid changes relative to wildtype Cry2Ab (K to R at position 36 and M to T at position 241 of GenBankAccession No. M23724). In another specific embodiment, clones isolatedfrom one round of alteration can be used as template for further roundsof alteration (e.g., clones 38, 44, and 473R; see Sections 6.2 and 6.4).

Sequence alterations can be introduced by standard techniques such asdirected molecular evolution techniques e.g., DNA shuffling methods (seee.g., Christians et al., 1999, Nature Biotechnology 17:259-264; Crameriet al., 1998, Nature, 391:288-291; Crameri, et al., 1997, NatureBiotechnology 15:436-438; Crameri et al., 1996, Nature Biotechnology14:315-319; Stemmer, 1994, Nature 370:389-391; Stemmer et al., 1994,Proc. Natl. Acad. Sci., 91:10747-10751; U.S. Pat. Nos. 5,605,793;6,117,679; 6,132,970; 5,939,250; 5,965,408; 6,171,820; InternationalPublication Nos. WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO00/42651; and WO 01/75767); site directed mutagenesis (see e.g., Kunkel,1985, Proc. Natl. Acad. Sci., 82:488-492; Oliphant et al., 1986, Gene44:177-183); oligonucleotide-directed mutagenesis (see e.g.,Reidhaar-Olson et al., 1988, Science 241:53-57); chemical mutagenesis(see e.g., Eckert et al., 1987, Mutat. Res. 178:1-10); error prone PCR(see e.g., Caldwell & Joyce, 1992, PCR Methods Applic. 2:28-33); andcassette mutagenesis (see e.g., Arkin et al., Proc. Natl. Acad. Sci.,1992, 89:7871-7815); (see generally, e.g., Arnold, 1993, Curr. OpinionBiotechnol. 4:450-455; Ling et al., 1997, Anal. Biochem., 254(2):157-78;Dale et al., 1996, Methods Mol. Biol. 57:369-74; Smith, 1985, Ann. Rev.Genet. 19:423-462; Botstein et al., 1985, Science, 229:1193-1201;Carter, 1986, Biochem. J. 237:1-7; Kramer et al., 1984, Cell 38:879-887;Wells et al., 1985, Gene 34:315-323; Minshull et al., 1999, CurrentOpinion in Chemical Biology 3:284-290).

In one embodiment, DNA shuffling is used to create Cry2Ax-derivednucleotides. DNA shuffling can be accomplished in vitro, in vivo, insilico, or a combination thereof. In silico methods of recombination canbe effected in which genetic algorithms are used in a computer torecombine sequence strings which correspond to homologous (or evennon-homologous) nucleic acids. The resulting recombined sequence stringsare optionally converted into nucleic acids by synthesis of nucleicacids which correspond to the recombined sequences, e.g., in concertwith oligonucleotide synthesis gene reassembly techniques. This approachcan generate random, partially random or designed alterations. Manydetails regarding in silico recombination, including the use of geneticalgorithms, genetic operators and the like in computer systems, combinedwith generation of corresponding nucleic acids as well as combinationsof designed nucleic acids (e.g., based on cross-over site selection) aswell as designed, pseudo-random or random recombination methods aredescribed in the art (see e.g., International Publication Nos. WO00/42560 and WO 00/42559).

In another embodiment, targeted mutagenesis is used to createCry2Ax-derived nucleotides by choosing particular nucleotide sequencesor positions of the Cry2Ax or related nucleic acid for alteration. Suchtargeted mutations can be introduced at any position in the nucleicacid. For example, one can make nucleotide substitutions leading toamino acid substitutions at “non-essential” or “essential” amino acidresidues. A “non-essential” amino acid residue is a residue that can bealtered from the wild-type sequence without altering the biologicalactivity, whereas an “essential” amino acid residue is required for atleast one biological activity of the polypeptide. For example, aminoacid residues that are not conserved or only semi-conserved amonghomologs of various species may be non-essential for activity.Alternatively, amino acid residues that are conserved among the homologsof various species may be essential for activity.

Such targeted mutations can be conservative or non-conservative. A“non-conservative amino acid substitution” is one in which the aminoacid residue is replaced with an amino acid residue having a dissimilarside chain. Families of amino acid residues having similar side chainshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid, asparagine, glutamine),uncharged polar side chains (e.g., glycine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan), β-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Alternatively or in addition to non-conservative amino acid residuesubstitutions, such targeted mutations can be conservative. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Following mutagenesis, the encoded protein can be expressedrecombinantly and the activity of the protein can be determined.

In another embodiment, random mutagenesis is used to createCry2Ax-derived nucleotides. Mutations can be introduced randomly alongall or part of the coding sequence (e.g., by saturation mutagenesis). Incertain embodiments, nucleotide sequences encoding other relatedpolypeptides that have similar domains, structural motifs, active sites,or that align with a portion of the Cry2Ax of the invention withmismatches or imperfect matches, can be used in the mutagenesis processto generate diversity of sequences.

It should be understood that for each mutagenesis step in some of thetechniques mentioned above, a number of iterative cycles of any or allof the steps may be performed to optimize the diversity of sequences.The above-described methods can be used in combination in any desiredorder. In many instances, the methods result in a pool of alterednucleic acid sequences or a pool of recombinant host cells comprisingaltered nucleic acid sequences. The altered nucleic acid sequences orhost cells expressing an altered nucleic acid sequence with the desiredcharacteristics can be identified by screening with one or more assaysknown in the art. The assays may be carried out under conditions thatselect for polypeptides possessing the desired physical or chemicalcharacteristics. The alterations in the nucleic acid sequence can bedetermined by sequencing the nucleic acid molecule encoding the alteredpolypeptide in the clones.

Additionally, Cry2Ax and Cry2Ax-derived nucleic acid molecules can becodon optimized, either wholly or in part. Because any one amino acid(except for methionine) is encoded by a number of codons (Table 2), thesequence of the nucleic acid molecule may be changed without changingthe encoded amino acid. Codon optimization is when one or more codonsare altered at the nucleic acid level such that the amino acids are notchanged but expression in a particular host organism is increased. Thosehaving ordinary skill in the art will recognize that tables and otherreferences providing preference information for a wide range oforganisms are available in the art.

5.4 Methods of Assaying Insecticidal Activity

As used herein, the term “insecticidal activity” refers to the abilityof a polypeptide to decrease or inhibit insect feeding and/or toincrease insect mortality upon ingestion of the polypeptide. Althoughany insect may be effected, preferably insects of the Lepidoptera andDiptera orders of insects are affected.

A variety of assays can be used to determine whether a particularpolypeptide of the invention has insecticidal activity and, if so, towhat degree. Generally, an insect pest is provided a polypeptide of theinvention in any form that can be ingested. The reaction of the insectpest to ingestion of the polypeptide of the invention is observed (e.g.,for about one to three days). A decrease or inhibition of feeding and/oran increase in insect pest mortality after ingestion of the polypeptideof the invention are indicators of insecticidal activity. A polypeptideof the invention with unknown insecticidal activity should be comparedto a positive and/or negative control to assess more accurately theoutcome of the assay.

In one embodiment, a polypeptide of the invention is purified (either insoluble form or in crystal form) and added to the insect diet.

In another embodiment, a polypeptide of the invention is expressed in arecombinant microbe (e.g., E. coli). The recombinant microbe is feddirectly to the insect pests (see Moellenbeck et al., 2001, Nat.Biotechnol. 19:668).

In another embodiment, the polypeptide of the invention is expressed ina plant and the plant is fed to the insect pest. Following theincubation period, the feeding activity of the insect pest can bedetermined by visual observation (e.g., of approximate fraction of leafarea remaining) or video capture (e.g., number of pixels in a leaf arearemaining) of the plant parts that would normally have been eaten by theinsect pest. In a specific embodiment, expression of the polypeptide ofthe invention in the plant is transient. In such embodiments, a nucleicacid encoding a polypeptide of the invention is cloned into a plantexpression vector and transfected into Agrobacterium tumefaciens. Thetransformed bacteria is co-cultivated with a leaf from N. benthamianaand, using forced infiltration, the leaf expresses the polypeptide ofthe invention. However, expression of the polypeptide is variablebetween leaf co-cultures. In another specific embodiment, expression ofthe polypeptide of the invention in the plant is stable. In suchembodiments, a transgenic plant is made that expresses a polypeptide ofthe invention.

In another embodiment, insecticidal activity of a polypeptide of theinvention can be assayed by measuring cell death and/or cell growthusing cultured cells. Such assays typically involve the use of culturedinsect cells that are susceptible to the particular toxin beingscreened, or cells that express a receptor for the particular toxin,either naturally or as a result of expression of a heterologous gene.Thus, in addition to insect cells, mammalian, bacterial, and yeast cellsare among those cells useful in the in vitro assays. In vitro bioassayswhich measure toxicity against cultured cells are described in the art(e.g., Johnson, 1994, J. Invertebr. Pathol. 63:123-129).

In another embodiment, insecticidal activity of a polypeptide of theinvention can be assayed by measuring pore formation in targetinsect-derived midgut epithelial membrane vesicles (Juttner and Ebel,1998, Biochim. Biophys. Acta 1370:51-63; English et al., 1991, InsectBiochem. 21:177-184). Such an assay may constitute toxin conditionalrelease of a ligand activated substrate from the lumen of the membranevesicles. This requires that the ligand be on the outside of thevesicle. Alternatively the reverse scenario may be utilized whereby theligand is in the vesicle lumen and the ready to be activated substrateis located on the outside of the vesicle. The higher the toxin activitythe greater the number or size of pores formed.

5.5 Methods of Enhancing Insect Resistance in Plants

The present invention provides methods of enhancing plant resistance toinsect pests including, but not limited to, members of the Helicoverpassp. (e.g., Helicoverpa Zea) and/or Spodoptera ssp. (e.g., Spodopterexigua) through the use of Cry2 related insecticidal polypeptides. Anymethod known in the art can be used to cause the insect pests to ingestone or more polypeptides of the invention during the course of feedingon the plant. As such, the insect pest will ingest insecticidal amountsof the one or more polypeptides of the invention and may discontinuefeeding on the plant. In some embodiments, the insect pest is killed byingestion of the one or more polypeptides of the invention. In otherembodiments, the insect pests are inhibited or discouraged from feedingon the plant without being killed.

In one embodiment, transgenic plants can be made to express one or morepolypeptides of the invention (see generally Section 5.7 for methods oftransgenic plant production). The transgenic plant may express the oneor more polypeptides of the invention in all tissues (e.g., globalexpression). Alternatively, the one or more polypeptides of theinvention may be expressed in only a subset of tissues (e.g., tissuespecific expression), preferably those tissues consumed by the insectpest. Polypeptides of the invention can be expressed constitutively inthe plant or be under the control of an inducible promoter.

In another embodiment, a composition comprising one or more polypeptidesof the invention can be applied externally to a plant susceptible to theinsect pests. External application of the composition includes directapplication to the plant, either in whole or in part, and/or indirectapplication, e.g., to the environment surrounding the plant such as thesoil. The composition can be applied by any method known in the artincluding, but not limited to, spraying, dusting, sprinkling, or thelike. In general, the composition can be applied at any time duringplant growth. One skilled in the art can use methods known in the art todetermine empirically the optimal time for administration of thecomposition. Factors that affect optimal administration time include,but are not limited to, the type of susceptible plant, the type ofinsect pest, which one or more polypeptides of the invention areadministered in the composition.

The composition comprising one or more polypeptides of the invention maybe substantially purified polypeptides, a cell suspension, a cellpellet, a cell supernatant, a cell extract, or a spore-crystal complexof Bacillus thuringiensis cells (see generally Section 5.6 forrecombinant polypeptide synthesis techniques). The compositioncomprising one or more polypeptides of the invention may be in the formof a solution, an emulsion, a suspension, or a powder. Liquidformulations may be aqueous or non-aqueous based and may be provided asfoams, gels, suspensions, emulsifiable concentrates, or the like. Theformulations may include agents in addition to the one or morepolypeptides of the invention. For example, compositions may furthercomprise spreader-sticker adjuvants, stabilizing agents, otherinsecticidal additives, diluents, agents that optimize the rheologicalproperties or stability of the composition, such as, for example,surfactants, emulsifiers, dispersants, or polymers.

In another embodiment, recombinant hosts that express one or morepolypeptides of the invention are applied on or near a plant susceptibleto attack by an insect pest. The recombinant hosts include, but are notlimited to, microbial hosts and insect viruses that have beentransformed with and express one or more nucleic acid molecules (andthus polypeptides) of the invention. In some embodiments, therecombinant host secretes the polypeptide of the invention into itssurrounding environment so as to contact an insect pest. In otherembodiments, the recombinant hosts colonize one or more plant tissuessusceptible to insect infestation.

5.6 Recombinant Expression

Nucleic acid molecules and polypeptides of the invention can beexpressed recombinantly using standard recombinant DNA and molecularcloning techniques that are well known in the art (e.g., Sambrook,Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual; ColdSpring Harbor Laboratory Press: Cold Spring Harbor, 1989). Additionally,recombinant DNA techniques may be used to create nucleic acid constructssuitable for use in making transgenic plants (see Section 5.7).

Accordingly, an aspect of the invention pertains to vectors, preferablyexpression vectors, comprising a nucleic acid molecule of the invention,or a variant thereof. As used herein, the term “vector” refers to apolynucleotide capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe introduced. Another type of vector is a viral vector, whereinadditional DNA segments can be introduced into the viral genome.

Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal vectors). Other vectors(e.g., non-episomal vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids (vectors).However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses).

The recombinant expression vectors of the invention comprise a nucleicacid molecule of the invention in a form suitable for expression of thenucleic acid molecule in a host cell. This means that the recombinantexpression vectors include one or more regulatory sequences, selected onthe basis of the host cells to be used for expression, which is operablyassociated with the polynucleotide to be expressed. Within a recombinantexpression vector, “operably associated” is intended to mean that thenucleotide sequence of interest is linked to the regulatory sequence(s)in a manner which allows for expression of the nucleotide sequence(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). The term “regulatorysequence” is intended to include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described in the art (e.g., Goeddel, GeneExpression Technology: Methods in Enzymology, 1990, Academic Press, SanDiego, Calif.). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcells and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences). Itwill be appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, thearea of the organism in which expression is desired, etc. The expressionvectors of the invention can be introduced into host cells to therebyproduce proteins or peptides, including fusion proteins or peptides,encoded by nucleic acids molecules as described herein.

In some embodiments, isolated nucleic acids which serve as promoter orenhancer elements can be introduced in the appropriate position(generally upstream) of a non-heterologous form of a polynucleotide ofthe present invention so as to up or down regulate expression of apolynucleotide of the present invention. For example, endogenouspromoters can be altered in vivo by mutation, deletion, and/orsubstitution (see, U.S. Pat. No. 5,565,350; International PatentApplication No. PCT/US93/03868), or isolated promoters can be introducedinto a plant cell in the proper orientation and distance from a cognategene of a polynucleotide of the present invention so as to control theexpression of the gene. Gene expression can be modulated underconditions suitable for plant growth so as to alter the totalconcentration and/or alter the composition of the polypeptides of thepresent invention in plant cell. Thus, the present invention providescompositions, and methods for making heterologous promoters and/orenhancers operably linked to a native, endogenous (i.e.,non-heterologous) form of a polynucleotide of the present invention.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

The recombinant expression vectors of the invention can be designed forexpression of a polypeptide of the invention in prokaryotic (e.g.,Enterobacteriaceae, such as Escherichia; Bacillaceae; Rhizoboceae, suchas Rhizobium and Rhizobacter; Spirillaceae, such as photobacterium;Zymomonas; Serratia; Aeromonas; Vibrio; Desulfovibrio; Spirillum;Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter;Azotobacteraceae and Nitrobacteraceae) or eukaryotic cells (e.g., insectcells using baculovirus expression vectors, yeast cells, plant cells, ormammalian cells) (see Goeddel, supra. For a discussion on suitable hostcells). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors comprising constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve at least three purposes: 1) to increaseexpression of the recombinant protein; 2) to increase the solubility ofthe recombinant protein; and/or 3) to aid in the purification of therecombinant protein by acting as a ligand in affinity purification.Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the fusion moiety and the recombinantprotein to enable separation of the recombinant protein from the fusionmoiety subsequent to purification of the fusion protein. Such enzymes,and their cognate recognition sequences, include Factor Xa, thrombin andenterokinase. Typical fusion expression vectors include pGEX (PharmaciaBiotech Inc; Smith and Johnson, 1988, Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari et al., 1987, EMBO J. 6:229-234), pMFa (Kurjanand Herskowitz, 1982, Cell 30:933-943), pJRY 88 (Schultz et al., 1987,Gene 54:113-123), pYES2 (Invitrogen Corp., San Diego, Calif.), and pPicZ(Invitrogen Corp., San Diego, Calif.).

Alternatively, the expression vector is a baculovirus expression vector.

Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.,1983, Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow andSummers, 1989, Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin plant cells using a plant expression vector including, but notlimited to, tobacco mosaic virus and potato virus expression vectors.

Other suitable expression systems for both prokaryotic and eukaryoticcells are known in the art (see, e.g., chapters 16 and 17 of Sambrook etal. 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.).

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-specific, inducible, orother promoters for expression in the host organism.

A “tissue-specific promoter” may direct expression of nucleic acids ofthe present invention in a specific tissue, organ or cell type.Tissue-specific promoters can be inducible. Similarly, tissue-specificpromoters may only promote transcription within a certain time frame ordevelopmental stage within that tissue. Other tissue specific promotersmay be active throughout the life cycle of a particular tissue. One ofordinary skill in the art will recognize that a tissue-specific promotermay drive expression of operably linked sequences in tissues other thanthe target tissue. Thus, as used herein, a tissue-specific promoter isone that drives expression preferentially in the target tissue or celltype, but may also lead to some expression in other tissues as well. Anumber of tissue-specific promoters can be used in the presentinvention. With the appropriate promoter, any organ can be targeted,such as shoot vegetative organs/structures (e.g. leaves, stems andtubers), roots, flowers and floral organs/structures (e.g. bracts,sepals, petals, stamens, carpels, anthers and ovules), seed (includingembryo, endosperm, and seed coat) and fruit. For instance, promotersthat direct expression of nucleic acids in leaves, roots or flowers areuseful for enhancing resistance to pests that infect those organs. Forexpression of a polynucleotide of the present invention in the aerialvegetative organs of a plant, photosynthetic organ-specific promoters,such as the RBCS promoter (Khoudi et al., Gene 197:343, 1997), can beused. Root-specific expression of polynucleotides of the presentinvention can be achieved under the control of a root-specific promoter,such as, for example, the promoter from the ANR1 gene (Zhang and Forde,Science, 279:407, 1998). Other exemplary promoters include theroot-specific glutamine synthetase gene from soybean (Hirel et al.,1992, Plant Molecular Biology 20:207-218) and the root-specific controlelement in the GRP 1.8 gene of French bean (Keller et al., 1991, ThePlant Cell 3:1051-1061).

A “constitutive promoter” is defined as a promoter which will directexpression of a gene in all tissues and are active under mostenvironmental conditions and states of development or celldifferentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcription initiation region, the1′- or 2′-promoter derived from T-DNA of Agrobacterium tumafaciens, andother transcription initiation regions from various plant genes known tothose of ordinary skill in the art. Such genes include for example,ACT11 from Arabidopsis (Huang et al. 1996, Plant Mol. Biol. 33:125-139),Cat3 from Arabidopsis (GenBank Accession No. U43147, Zhong et al., 1996,Mol. Gen. Genet. 251:196-203), the gene encoding stearoyl-acyl carrierprotein desaturase from Brassica napus (Genbank Accession No. X74782,Solocombe et al. 1994, Plant Physiol. 104:1167-1176), GPc1 from maize(GenBank Accession No. X15596, Martinez et al., 1989, J. Mol. Biol.208:551-565), and Gpc2 from maize (GenBank Accession No. U45855,Manjunath et al., 1997, Plant Mol. Biol. 33:97-112). Any strong,constitutive promoter, such as the CaMV 35S promoter, can be used forthe expression of polynucleotides of the present invention throughoutthe plant.

The term “inducible promoter” refers to a promoter that is under preciseenvironmental or developmental control. Examples of environmentalconditions that may effect transcription by inducible promoters includeanaerobic conditions, elevated temperature, the presence of light, orspraying with chemicals/hormones.

Suitable constitutive promoters for use in a plant host cell include,for example, the core promoter of the Rsyn7 promoter and other relatedconstitutive promoters (International Publication No. WO 99/43838 andU.S. Pat. No. 6,072,050); the core CaMV 35S promoter (Odell et al.,1985, Nature 313:810-812); rice actin (McElroy et al., 1990, Plant Cell2:163-171); ubiquitin (Christensen et al., 1989, Plant Mol. Biol.12:619-632 and Christensen et al., 1992, Plant Mol. Biol. 18:675-689);pEMU (Last et al., 1991, Theor. Appl. Genet. 81:581-588); MAS (Velten etal., 1984, EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026),and the like (e.g., U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121;5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

Accordingly, the present invention provides a host cell having anexpression vector comprising a nucleic acid of the invention, or avariant thereof. A host cell can be any prokaryotic (e.g., E. coli,Bacillus thuringiensis) or eukaryotic cell (e.g., insect cells, yeast orplant cells). The invention also provides a method for expressing anucleic acid of the invention thus making the encoded polypeptidecomprising the steps of i) culturing a cell comprising a nucleic acidmolecule of the invention under conditions that allow production of theencoded polypeptide; and ii) isolating the expressed polypeptide.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid molecules into a host cell, including calcium phosphate or calciumchloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in the art (e.g., Sambrook, et al.supra.).

5.7 Production of Transgenic Plants

Any method known in the art can be used for transforming a plant orplant cell with a nucleic acid molecule of the present invention.Nucleic acid molecules can be incorporated into plant DNA (e.g., genomicDNA or chloroplast DNA) or be maintained without insertion into theplant DNA (e.g., through the use of artificial chromosomes). Suitablemethods of introducing nucleic acid molecules into plant cells includemicroinjection (Crossway et al., 1986, Biotechniques 4:320-334);electroporation (Riggs et al., 1986, Proc. Natl. Acad. Sci.83:5602-5606; D'Halluin et al., 1992, Plant Cell 4:1495-1505);Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and5,981,840, Osjoda et al., 1996, Nature Biotechnology 14:745-750; Horschet al., 1984, Science 233:496-498, Fraley et al., 1983, Proc. Natl.Acad. Sci. 80:4803, and Gene Transfer to Plants, Potrykus, ed.,Springer-Verlag, Berlin 1995); direct gene transfer (Paszkowski et al.,1984, EMBO J. 3:2717-2722); ballistic particle acceleration (U.S. Pat.Nos. 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes et al., 1995,“Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment, in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg and Phillips, Springer-Verlag, Berlin; and McCabeet al., 1988, Biotechnology 6:923-926); virus-mediated transformation(U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and5,316,931); pollen transformation (De Wet et al., 1985, in TheExperimental Manipulation of Ovule Tissues, ed. Chapman et al., Longman,New York, pp. 197-209); Lec 1 transformation (U.S. patent applicationSer. No. 09/435,054; International Publication No. WO 00/28058);whisker-mediated transformation (Kaeppler et al., 1990, Plant CellReports 9:415-418; Kaeppler et al., 1992, Theon. Appl. Genet.84:560-566); and chloroplast transformation technology (Bogorad, 2000,Trends in Biotechnology 18: 257-263; Ramesh et al., 2004, Methods Mol.Biol. 274:301-7; Hou et al., 2003, Transgenic Res. 12:111-4; Kindle etal., 1991, Proc. Natl. Acad. Sci. 88:1721-5; Bateman and Purton, 2000,Mol Gen Genet. 263:404-10; Sidorov et al., 1999, Plant J. 19:209-216).

The choice of transformation protocols used for generating transgenicplants and plant cells can vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Examples oftransformation protocols particularly suited for a particular plant typeinclude those for: potato (Tu et al., 1998, Plant Molecular Biology37:829-838; Chong et al., 2000, Transgenic Research 9:71-78); soybean(Christou et al., 1988, Plant Physiol. 87:671-674; McCabe et al., 1988,BioTechnology 6:923-926; Finer and McMullen, 1991, In Vitro Cell Dev.Biol. 27P:175-182; Singh et al., 1998, Theor. Appl. Genet. 96:319-324);maize (Klein et al., 1988, Proc. Natl. Acad. Sci. 85:4305-4309; Klein etal., 1988, Biotechnology 6:559-563; Klein et al., 1988, Plant Physiol.91:440-444; Fromm et al., 1990, Biotechnology 8:833-839; Tomes et al.,1995, “Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment,” in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg (Springer-Verlag, Berlin)); cereals (Hooykaas-VanSlogteren et al., 1984, Nature 311:763-764; U.S. Pat. No. 5,736,369).

In some embodiments, more than one construct is used for transformationin the generation of transgenic plants and plant cells. Multipleconstructs may be included in cis or trans positions. In preferredembodiments, each construct has a promoter and other regulatorysequences.

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and thus the desired phenotype.Such regeneration techniques rely on manipulation of certainphytohormones in a tissue culture growth medium, typically relying on abiocide and/or herbicide marker that has been introduced together withthe desired nucleotide sequences. Plant regeneration from culturedprotoplasts is described in the art (e.g., Evans et al., ProtoplastsIsolation and Culture, Handbook of Plant Cell Culture, pp. 124-176,MacMillilan Publishing Company, New York, 1983; and Binding,Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, BocaRaton, 1985). Regeneration can also be obtained from plant callus,explants, organs, or parts thereof. Such regeneration techniques arealso described in the art (e.g., Klee et al. 1987, Ann. Rev. of PlantPhys. 38:467-486).

The term “plant” includes whole plants, shoot vegetativeorgans/structures (e.g. leaves, stems and tubers), roots, flowers andfloral organs/structures (e.g. bracts, sepals, petals, stamens, carpels,anthers and ovules), seed (including embryo, endosperm, and seed coat)and fruit (the mature ovary), plant tissue (e.g. vascular tissue, groundtissue, and the like) and cells (e.g. guard cells, egg cells, trichomesand the like), and progeny of same. The class of plants that can be usedin methods of the present invention includes the class of higher andlower plants amenable to transformation techniques, includingangiosperms (monocotyledonous and dicotyledonous plants), gymnosperms,ferns, and multicellular algae. Plants of a variety of ploidy levels,including aneuploid, polyploid, diploid, haploid and hemizygous plantsare also included.

The nucleic acid molecules of the invention can be used to conferdesired traits on essentially any plant. Thus, the invention has useover a broad range of plants, including species from the genera Agrotis,Allium, Ananas, Anacardium, Apium, Arachis, Asparagus, Athamantha,Atropa, Avena, Bambusa, Beta, Brassica, Bromus, Browaalia, Camellia,Cannabis, Carica, Ceratonia. Cicer, Chenopodium, Chicorium, Citrus,Citrullus, Capsicum, Carthamus, Cocos, Coffea, Coix, Cucumis, Cucurbita,Cynodon, Dactylis, Datura, Daucus, Dianthus, Digitalis, Dioscorea,Elaeis, Eliusine, Euphorbia, Festuca, Ficus, Fragaria, Geranium,Glycine, Graminae, Gossypium, Helianthus, Heterocallis, Hevea, Hibiscus,Hordeum, Hyoscyamus, Ipomoea, Lactuca, Lathyrus, Lens, Lilium, Linum,Lolium, Lotus, Lupinus, Lycopersicon, Macadamia, Macrophylla, Malus,Mangifera, Manihot, Majorana, Medicago, Musa, Narcissus, Nemesia,Nicotiana, Onobrychis, Olea, Olyreae, Oryza, Panicum, Panicum, Panieum,Pannisetum, Pennisetum, Petunia, Pelargonium, Persea, Pharoideae,Phaseolus, Phleum, Picea, Poa, Pinus, Pistachia, Pisum, Populus,Pseudotsuga, Pyrus, Prunus, Pseutotsuga, Psidium, Quercus, Ranunculus,Raphanus, Ribes, Ricinus, Rhododendron, Rosa, Saccharum, Salpiglossis,Secale, Senecio, Setaria, Sequoia, Sinapis, Solanum, Sorghum,Stenotaphrum, Theobromus, Trigonella, Trifolium, Trigonella, Triticum,Tsuga, Tulipa, Vicia, Vitis, Vigna, and Zea.

In specific embodiments, transgenic plants are maize, potato, rice,soybean or cotton plants.

Transgenic plants may be grown and pollinated with either the sametransformed strain or different strains. Two or more generations of theplants may be grown to ensure that expression of the desired nucleicacid molecule, polypeptide and/or phenotypic characteristic is stablymaintained and inherited. One of ordinary skill in the art willrecognize that after the nucleic acid molecule of the present inventionis stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

5.8 Determination of Expression in Transgenic Plants

Any method known in the art can be used for determining the level ofexpression in a plant of a nucleic acid molecule of the invention orpolypeptide encoded therefrom. For example, the expression level in aplant of a polypeptide encoded by a nucleic acid molecule of theinvention can be determined by immunoassay, quantitative gelelectrophoresis, etc. Additionally, the expression level in a plant of apolypeptide encoded by a nucleic acid molecule of the invention can bedetermined by the degree to which the plant phenotype is altered. In aspecific embodiment, enhanced insect resistance is the phenotype to beassayed.

As used herein, “enhanced insect resistance” refers to increasedresistance of a transgenic plant expressing a polypeptide of theinvention to consumption and/or infestation by an insect pest ascompared to a plant not expressing a polypeptide of the invention.Enhanced resistance can be measured in a number of ways. In oneembodiment, enhanced resistance is measured by decreased damage to aplant expressing a polypeptide of the invention as compared to a plantnot expressing a polypeptide of the invention after the same period ofinsect incubation. Insect damage can be assessed visually. For examplein cotton plants, damage after infestation can be measured by lookingdirectly at cotton plant bolls for signs of consumption by insects. Inanother embodiment, enhanced resistance is measured by increased cropyield from a plant expressing a polypeptide of the invention as comparedto a plant not expressing a polypeptide of the invention after the sameperiod of insect incubation. In particular embodiments, the insect pestare from the orders Lepidoptera and/or Diptera.

Determinations can be made using whole plants, tissues thereof, or plantcell culture.

The Sequence Listing that is 0.97 MB and was created on Feb. 24, 2005and was submitted to the PCT authorities in the International Phase, isincorporated by reference in its entirety.

The contents of all published articles, books, reference manuals andabstracts cited herein, are hereby incorporated by reference in theirentirety to more fully describe the state of the art to which theinvention pertains.

As various changes can be made in the above-described subject matterwithout departing from the scope and spirit of the present invention, itis intended that all subject matter contained in the above description,and/or defined in the appended claims, be interpreted as descriptive andillustrative of the present invention. Modifications and variations ofthe present invention are possible in light of the above teachings.

6. EXAMPLES 6.1 Example 1 Primary Insect Screening

Primary insect screening identified Bt cultures from a biodiversitycollection having activity against Helicoverpa spp. The screening wasconducted with spore-crystal complex samples at one high dose againstHelicoverpa zea neonate larvae.

Spore-crystal complex samples were prepared in deep-well productionplates containing 1 ml CYS sporulation medium (Yamamoto, 1990,Analytical Chemistry of Bacillus thuringiensis 432:46-60). Theproduction plates were inoculated with 10 μl seed cultures that had beenkept frozen at −80° C. and incubated at 30° C., 350 rpm for 3 days untilmost cultures sporulated and released free spores and crystals. Theplates were centrifuged at 4000 rpm, for 40 min to precipitate spores,crystals and unlysed cells. The precipitated spore-crystal complex wassuspended in 1.2 ml 15 mM potassium acetate containing 100 μg lysozymeand incubated at 30° C., 250 rpm for 16 h to ensure full sporulation andcell lysis. After the 16-hr incubation, the spore-crystal complex wascollected by centrifugation and suspended in 15 mM potassium acetate.

This potassium acetate step was repeated once. The final spore-crystalsuspension was made in 1 ml 15 mM potassium acetate and used to screenfor H. zea activity.

Insect screening was done in shallow 96-well plates containing 150 μlartificial insect diet in each well. 20 μl of spore-crystal suspensionwas placed on the insect diet. About 5 neonate larvae were placed ineach well. The insect assay plates were incubated at 29° C. for 3 days.Insect responses to Bt crystals included feeding inhibition andmortality. About 400 cultures showed substantial mortality and weretherefore identified as positive. Cry2Ax was among the positives.

6.2 Example 2 DNA Shuffling to Isolate Cry2Ax-Derived Polypeptides

Starting with the Cry2Ax polypeptide (SEQ ID NO:2) and the Cry2Ab*polypeptide (Cry2Ab* has 2 amino acid changes relative to wild typeCry2Ab; K to R at position 36 and M to T at position 241 of GenBankAccession No. M23724), synthetic DNA templates were created for DNAshuffling. Using a phylogenetic comparison of Cry2Ab (Gen Bank AccessionNo. M23724) and Cry2Ax (SEQ ID NO:2) a library was created that variedthe 40 amino acid positions (see Section 5.1) that were differentbetween these two polypeptide sequences. Shuffled DNA libraries werecreated using oligonucleotide directed shuffling with the synthetic geneencoding Cry2Ax acting as the parental DNA template. The PCR DNAlibraries were cloned into pMAXY3219 by replacing the Cry2Ab* gene. Thetoxin clones were built such that they are expressed as a fusion to theE. coli maltose binding protein (MBP). Primary insect screeningidentified cultures active against Helicoverpa spp. The screening wasconducted by incorporating a small amount of E. coli culture expressingthe MBP::Cry2Ax-derived polypeptide fusion at one low dose in artificialdiet followed by infestation with H. zea larvae. Screening was done inshallow 96-well plates containing 150 μl artificial insect diet in eachwell. ˜0.5 μl of MBP::Cry2Ax-derived polypeptide fusion expressingculture was incorporated into the insect diet. About 5H. zea neonatelarvae were placed in each well. The insect assay plates were incubatedat 29° C. for 4 days. Insect responses to the E. coli samples includedfeeding inhibition and mortality. Those samples causing severe stuntingor death to the larvae were re-arrayed for further analysis.

Screening of this first round library led to the discovery of severalclones improved insecticidal activity relative to Cry2Ax and Cry2Ab*. Inparticular, clones 38 (D_(—)500503480) (SEQ ID NO:4) and 44(D_(—)500503970) (SEQ ID NO:6) were found to be highly active whenexpressed (data not shown). These clones were therefore chosen for asuccessive round of DNA shuffling.

For the second round of shuffling, parent DNA templates from clones 38(D_(—)500503480) (SEQ ID NO:4) and 44 (D_(—)500503970) (SEQ ID NO:6)were PCR amplified in the presence of uracil and then fragmented withuracil N-glycosylase. The fragmented templates were then mixed,reassembled before recombinant templates were amplified by PCR. Alibrary of these shuffled templates was created in pMAXY3219 asdescribed above. The sequence of some of the clones isolated from thefirst and second rounds of shuffling is shown in Table 3 indicating theamino acid residues that were changed.

In order to further diversify one of the top performing 2^(nd) Roundhits, clone 473R (D_(—)501037677) (SEQ ID NO:18), the first 46 aminoacid residues at the amino terminal region of the polypeptide weremodified to contain residues found in eight different Cry2 polypeptidesequences (i.e., Cry2Aa, Cry2Ab, Cry2Ac, Cry2Ad, Cry2Ae, Cry2Af, Cry2Ag,and Cry2Ax). In addition, two residues, I13 and D15, were substitutedwith conservative residues valine and glutamate, respectively (see Table4). The modified clone was termed clone 473N (SEQ ID NO:8).

6.3 Example 3 Activity of Cry2Ax-derived Polypeptides

Screening of the activities of the shuffled clones was carried out inseveral stages. Initially the clones were screened for high insecticidalactivity by providing a small amount of E. coli expressing a clonefusion protein into artificial diet for first instar H. zea larvae.Clones causing either complete death or complete stunting of the larvaewere chosen for further study. Those clones that demonstrated highinsecticidal activity were then used to create a new library in a plantexpression vector in Agrobacterium tumefaciens. The library was screenedby co-cultivating each clone in four replicates with N. benthamianaleaves (using forced infiltration of each respective culture), and thenfeeding each corresponding disk to a single 3^(rd) instar H. zea larvae.Following a 24-hour incubation period, the feeding activity wasdetermined by visual observation and expressed as an approximatefraction of leaf area remaining

The clones passing further repetition of the E. coli expression/dietincorporation assays were re-cloned individually into plant expressionvector pMAXY4384 and tested for efficacy in planta as described above. Afinal in planta activity assessment of the best hits from the E. coliexpression multi-tiered assay and the plant library approach is shown inFIG. 1. From this analysis several clones appeared to have increasedinsecticidal activity including 7K (D_S01000779) (SEQ ID NO:10), 15K(D_(—)500999080) (SEQ ID NO:12), 16K (D_(—)501000269) (SEQ ID NO:14),16R (D_(—)501037143) (SEQ ID NO:16), and 473R (D_(—)501037677) (SEQ IDNO:18).

6.4 Example 4 DNA Shuffling to Isolate Additional Cry2Ax-DerivedPolypeptides

Clones 44 (D_(—)500503970) (SEQ ID NO:6), 473R (D_S01037677) (SEQ ID NO:18) which were 1^(st) and 2^(nd) round shuffling hits as describedSection 6.2 and Cry2Ab* were used as templates for further shuffling.Using these templates and oligonucleotide directed shuffling, derivedpolypeptides were created having amino acid diversity from wild typeCry2 polypeptides (i.e., Cry2Ae and Cry2Ag) as well as computergenerated random conservative amino acid substitutions and randomsubstitutions within segments of certain structural loop regions. Theshuffled DNA libraries were cloned into pMAXY3219 by replacing theCry2Ab* gene. The toxin clones were built such that they were expressedas a fusion to the E. coli maltose binding protein (MBP). A summary ofthe isolated sequences is shown in Tables 5-7.

6.5 Example 5 Activity of Additional Cry2Ax-Derived Polypeptides

In order to assess the activity of the shuffled derived polypeptidesagainst the cotton pest Helicoverpa zea, high throughput screening usingartificial diet containing whole E. coli cells expressing a clone fusionprotein was performed as described supra. Clones having a high level ofactivity were further tested for in planta activity to confirm that thechanges made to each derived polypeptide did not negatively impact geneexpression or protein accumulation in plant cells. To initiate thisprocess, each Cry2Ax-derived polypeptide was cloned into anAgrobacterium tumefaciens based plant expression vector, transformedinto the host Agrobacterium strain and then arrayed into microtiterdishes. The hits were then screened by co-cultivating each in fourreplicates with N. benthamiana leaves (using forced infiltration of eachrespective culture), followed by feeding each corresponding disk to asingle 3^(rd) instar H. zea larvae. Following a 24-hour incubationperiod the feeding activity on each disc was determined by the visualcapture and analysis method as described supra. Some derivedpolypeptides from this process were improved compared to the parentalclones. One such clone is D_S01764701 (SEQ ID NO:134) that showedimproved activity over clone 44. Feeding assay results are shown forthree experiments in FIG. 2.

6.6 Example 6 Transgenic Plants Expressing Clone 44

Transgenic tobacco plants expressing clone 44 (D_S00503970) weregenerated by Agrobacterium-mediated transformation with glyphosateselection using binary vectors pMAXY5469 and pMAXY5471. These vectorscontain a dSVBV driven GAT gene and a dMMV driven clone 44 nucleic acidmolecule clone 44 (SEQ ID NO:5). pMAXY5469 differs from pMAXY5471 inthat it contains plastid targeting signal fused to the coding region ofclone 44 such that this toxin variant will accumulate in the plastidcompartment. Approximately 25 transformants were generated for eachconstruct. Leaf disks expressing clone 44 were placed on a bed of agarin a 48-well titer tray and then infested with either 3rd instarHelicoverpa zea larvae or 4th instar Spodoptera exigua larvae. Theleaves were incubated 24 hrs with the worms and then the larvae whichwere then removed and the leaf remaining was observed with video captureequipment for actual calculation of relative leaf area remaining (numberof pixels). Results using the top transformants for each vector areshown in FIG. 3A for the H. zea and FIG. 3B for the S. exigua. Eachtransgenic plant has 6 leaf disks taken for analysis as shown.

Expression of then clone 44 polypeptide in the transgenic tobacco plantsin the plastid (FIG. 4A) or in the cytoplasmic compartment (FIG. 4B) wasassayed by western blot using a polyclonal antibody directed to clone 44polypeptide. Lane numbers in FIG. 4 correspond to plant numbers in FIG.3.

The polyclonal antibody used in the western blot was prepared byimmunizing chickens with purified trypsin truncated clone 44 polypeptideand then purifying Cry2 specific antibodies using an affinity columnmade with trypsin truncated clone 44 polypeptide as the substrate.

The most obvious difference between the two types of transgenic plantsis that inhibition of S. exigua is much greater for theplastid-accumulated toxin (comparing right and left panels of FIG. 3B).These data in conjunction with the expression data (FIG. 4) showing thatplants harboring the T-DNA derived from 5469 (FIG. 4A) are capable ofproducing far more toxin than those of 5471 (FIG. 4B).

TABLE 1 Cry2Ax and Cry2Ax-derived sequences SEQ Clone name Type ID NOCry2Ax nucleic acid 1 Cry2Ax polypeptide 2 38 (D_S00503480) nucleic acid3 38 (D_S00503480) polypeptide 4 44 (D_S00503970) nucleic acid 5 44(D_S00503970) polypeptide 6 473N nucleic acid 7 473N polypeptide 8 7K(D_S01000779) nucleic acid 9 7K (D_S01000779) polypeptide 10 15K(D_S00999080) nucleic acid 11 15K (D_S00999080) polypeptide 12 16K(D_S01000269) nucleic acid 13 16K (D_S01000269) polypeptide 14 16R(D_S01037143) nucleic acid 15 16R (D_S01037143) polypeptide 16 473R(D_S01037677) nucleic acid 17 473R (D_S01037677) polypeptide 18D_S01466681 nucleic acid 19 D_S01466681 polypeptide 20 D_S01466770nucleic acid 21 D_S01466770 polypeptide 22 D_S01467219 nucleic acid 23D_S01467219 polypeptide 24 D_S01466712 nucleic acid 25 D_S01466712polypeptide 26 D_S01467003 nucleic acid 27 D_S01467003 polypeptide 28D_S01460229 nucleic acid 29 D_S01460229 polypeptide 30 D_S01459398nucleic acid 31 D_S01459398 polypeptide 32 D_S01464856 nucleic acid 33D_S01464856 polypeptide 34 D_S014657862 nucleic acid 35 D_S014657862polypeptide 36 D_S01458733 nucleic acid 37 D_S01458733 polypeptide 38D_S01457892 nucleic acid 39 D_S01457892 polypeptide 40 D_S01442158nucleic acid 41 D_S01442158 polypeptide 42 D_S01443366 nucleic acid 43D_S01443366 polypeptide 44 D_S01442132 nucleic acid 45 D_S01442132polypeptide 46 D_S01532970 nucleic acid 47 D_S01532970 polypeptide 48D_S01532041 nucleic acid 49 D_S01532041 polypeptide 50 D_S01611723nucleic acid 51 D_S01611723 polypeptide 52 D_S01561293 nucleic acid 53D_S01561293 polypeptide 54 D_S01561489 nucleic acid 55 D_S01561489polypeptide 56 D_S01561330 nucleic acid 57 D_S01561330 polypeptide 58D_S01570511 nucleic acid 59 D_S01570511 polypeptide 60 D_S01570809nucleic acid 61 D_S01570809 polypeptide 62 D_S01570568 nucleic acid 63D_S01570568 polypeptide 64 D_S01572168 nucleic acid 65 D_S01572168polypeptide 66 D_S01571315 nucleic acid 67 D_S01571315 polypeptide 68D_S01571875 nucleic acid 69 D_S01571875 polypeptide 70 D_S01572374nucleic acid 71 D_S01572374 polypeptide 72 D_S01572905 nucleic acid 73D_S01572905 polypeptide 74 D_S01572908 nucleic acid 75 D_S01572908polypeptide 76 D_S01561856 nucleic acid 77 D_S01561856 polypeptide 78D_S01573294 nucleic acid 79 D_S01573294 polypeptide 80 D_S01571529nucleic acid 81 D_S01571529 polypeptide 82 D_S01599948 nucleic acid 83D_S01599948 polypeptide 84 D_S01601459 nucleic acid 85 D_S01601459polypeptide 86 D_S01602925 nucleic acid 87 D_S01602925 polypeptide 88D_S01613034 nucleic acid 89 D_S01613034 polypeptide 90 D_S01614407nucleic acid 91 D_S01614407 polypeptide 92 D_S01631557 nucleic acid 93D_S01631557 polypeptide 94 D_S01633080 nucleic acid 95 D_S01633080polypeptide 96 D_S01632237 nucleic acid 97 D_S01632237 polypeptide 98D_S01633031 nucleic acid 99 D_S01633031 polypeptide 100 D_S01632121nucleic acid 101 D_S01632121 polypeptide 102 D_S01764500 nucleic acid103 D_S01764500 polypeptide 104 D_S01764502 nucleic acid 105 D_S01764502polypeptide 106 D_S01764505 nucleic acid 107 D_S01764505 polypeptide 108D_S01764533 nucleic acid 109 D_S01764533 polypeptide 110 D_S01764543nucleic acid 111 D_S01764543 polypeptide 112 D_S01764546 nucleic acid113 D_S01764546 polypeptide 114 D_S01764554 nucleic acid 115 D_S01764554polypeptide 116 D_S01764568 nucleic acid 117 D_S01764568 polypeptide 118D_S01764569 nucleic acid 119 D_S01764569 polypeptide 120 D_S01764577nucleic acid 121 D_S01764577 polypeptide 122 D_S01764642 nucleic acid123 D_S01764642 polypeptide 124 D_S01764643 nucleic acid 125 D_S01764643polypeptide 126 D_S01764680 nucleic acid 127 D_S01764680 polypeptide 128D_S01764685 nucleic acid 129 D_S01764685 polypeptide 130 D_S01764691nucleic acid 131 D_S01764691 polypeptide 132 D_S01764701 nucleic acid133 D_S01764701 polypeptide 134 D_S01764706 nucleic acid 135 D_S01764706polypeptide 136 D_S01764723 nucleic acid 137 D_S01764723 polypeptide 138D_S02847715 nucleic acid 139 D_S02847715 polypeptide 140 D_S01765051nucleic acid 141 D_S01765051 polypeptide 142 D_S01765068 nucleic acid143 D_S01765068 polypeptide 144 D_S01765100 nucleic acid 145 D_S01765100polypeptide 146 D_S01765063 nucleic acid 147 D_S01765063 polypeptide 148D_S01765119 nucleic acid 149 D_S01765119 polypeptide 150 D_S01765104nucleic acid 151 D_S01765104 polypeptide 152 D_S01765112 nucleic acid153 D_S01765112 polypeptide 154 D_S01765174 nucleic acid 155 D_S01765174polypeptide 156 D_S01765242 nucleic acid 157 D_S01765242 polypeptide 158D_S01765308 nucleic acid 159 D_S01765308 polypeptide 160 D_S01765221nucleic acid 161 D_S01765221 polypeptide 162 D_S01765254 nucleic acid163 D_S01765254 polypeptide 164 D_S01765231 nucleic acid 165 D_S01765231polypeptide 166 D_S01765255 nucleic acid 167 D_S01765255 polypeptide 168D_S01765377 nucleic acid 169 D_S01765377 polypeptide 170 D_S01765430nucleic acid 171 D_S01765430 polypeptide 172 D_S01765446 nucleic acid173 D_S01765446 polypeptide 174 D_S01765496 nucleic acid 175 D_S01765496polypeptide 176 D_S01764642 nucleic acid 177 D_S01764642 polypeptide 178D_S01766041 nucleic acid 179 D_S01766041 polypeptide 180 D_S01764706nucleic acid 181 D_S01764706 polypeptide 182 D_S01766073 nucleic acid183 D_S01766073 polypeptide 184 D_S01764643 nucleic acid 185 D_S01764643polypeptide 186 D_S01763985 nucleic acid 187 D_S01763985 polypeptide 188D_S01764668 nucleic acid 189 D_S01764668 polypeptide 190 D_S01764196nucleic acid 191 D_S01764196 polypeptide 192 D_S01764728 nucleic acid193 D_S01764728 polypeptide 194 D_S01764787 nucleic acid 195 D_S01764787polypeptide 196 D_S01764758 nucleic acid 197 D_S01764758 polypeptide 198D_S01764768 nucleic acid 199 D_S01764768 polypeptide 200 D_S01764860nucleic acid 201 D_S01764860 polypeptide 202 D_S01765018 nucleic acid203 D_S01765018 polypeptide 204 D_S01764947 nucleic acid 205 D_S01764947polypeptide 206 D_S01764934 nucleic acid 207 D_S01764934 polypeptide 208D_S01764968 nucleic acid 209 D_S01764968 polypeptide 210 D_S01765008nucleic acid 211 D_S01765008 polypeptide 212 D_S01764953 nucleic acid213 D_S01764953 polypeptide 214 D_S01764977 nucleic acid 215 D_S01764977polypeptide 216 D_S01765509 nucleic acid 217 D_S01765509 polypeptide 218D_S01765668 nucleic acid 219 D_S01765668 polypeptide 220 D_S01765621nucleic acid 221 D_S01765621 polypeptide 222 D_S01765693 nucleic acid223 D_S01765693 polypeptide 224 D_S01765687 nucleic acid 225 D_S01765687polypeptide 226 D_S01765765 nucleic acid 227 D_S01765765 polypeptide 228D_S01765932 nucleic acid 229 D_S01765932 polypeptide 230 D_S01766010nucleic acid 231 D_S01766010 polypeptide 232 D_S01766026 nucleic acid233 D_S01766026 polypeptide 234 D_S02838294 nucleic acid 235 D_S02838294polypeptide 236 D_S02838310 nucleic acid 237 D_S02838310 polypeptide 238D_S02838327 nucleic acid 239 D_S02838327 polypeptide 240 D_S02838328nucleic acid 241 D_S02838328 polypeptide 242 D_S02838330 nucleic acid243 D_S02838330 polypeptide 244 D_S02838454 nucleic acid 245 D_S02838454polypeptide 246 D_S02838470 nucleic acid 247 D_S02838470 polypeptide 248D_S02838478 nucleic acid 249 D_S02838478 polypeptide 250 D_S02838434nucleic acid 251 D_S02838434 polypeptide 252 D_S02838549 nucleic acid253 D_S02838549 polypeptide 254 D_S02838632 nucleic acid 255 D_S02838632polypeptide 256 D_S02838640 nucleic acid 257 D_S02838640 polypeptide 258D_S02838648 nucleic acid 259 D_S02838648 polypeptide 260 Cry2Abpolypeptide 261

TABLE 2 Codon Table Amino acids Codon Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

TABLE 3 Sequence of clones of interest isolated from DNA shuffling Aminoacid Parents 1^(st) Rd 2^(nd) Rd position 2Ab 2Ax 38 44 7K 15K 16K 16R473R 3 N H H H H H H H H Amino Acid Sequence 4 S N N N N N N N N 20 A VA A A A A A A 35 V I V V V V V V V 37 K E E E E E E E E 40 T M M M M M MM M 42 W W W W W W R W W 45 N D D D D D D D D 51 L V V V V L L V V 80 NN S N S S S S S 163 P T T P P P P P P 210 R Q R Q R R Q R R 211 D N N NN N N N N 212 Y H H H H H H H H 214 K R R R R R R R R 230 S T S T S S SS T 233 K R R R R R R R R 242 M M M M V M M M M 318 S T S S S S S S S319 N Q Q Q Q Q Q Q Q 330 S T T T T T T T T 347 I V V V V V V V V 355 S— S S S S S S S 356 P V P P P P P P P 358 N N T N T T T N N 362 N S S SS S S S S 386 E G G G G G G G G 388 V I V V V V V V V 389 A N A N A N NA N 447 E Q Q Q Q Q Q Q Q 452 A E E A E A A A E 461 A L A L A L L A A477 H Y Y Y Y Y Y Y Y 484 S T T S T S T T T 490 N E E E E E E E E 529 NS S S S S S S S 570 T S S T T S S S T 600 S D D S S S S S D 602 S T S SS S S S S 603 D N N D D D D D N 619 D E E D D E E D D 625 L F F F F F FF F 630 I L L L L L L L L 631 S P S P P S S P P

TABLE 4 Sequence comparison of wild type Cry2 polypeptides with clone473N Amino Modified acid Parents Clone position Cry2Aa Cry2Ab Cry2AcCry2Ad Cry2Ae Cry2Af Cry2Ag Cry2Ax 473R 473N 8 S S N S N S S S S N AminoAcid Sequence 9 S G G G G G E G G E 11 S T N N T T T T T N 13 S I T I II K I I V 15 S D H D D D G D D E 17 S Y H Y Y Y Y Y Y H 21 S A A V A A AA A V 22 S H H H H H H H H R 28 S Q E Q E Q E Q Q E 36 S Q E D R Q Q Q QE 37 S K K N K E K E E K 40 S T K M M M M M M T 44 S K R K R K R K K R45 S N T D T D T D D N 46 S N D N D N D N N D

TABLE 5 Sequence of clones of interest isolated from DNA shuffling usingCry2Ab* as parent Sample 1 3 4 7 9 10 11 12 13 14 20 25 26 27 28 29 3134 36 37 42 46 48 49 51 54 56 Cry2Ab* M N S N G R T T I C A F S F Q H ST Q R W N S L L I G Cry2Ab wt seq K D_S02838648 D P R R D_S02838640 RD_S02838632 R R P D_S02838470 R D_S02838434 R D_S02838328 R D_S02838327R D_S02838310 R D_S01766073 R D_S01766026 R D D_S01765621 R D_S01765509R D_S01765496 R R D_s01765255 R R D_S01765119 R D_S01765112 T R R V VD_S01765104 H R D_S01765063 R D_S01765008 R D_S01764977 R D_S01764947 LR D_S01764787 R D_S01764723 L R D_S01764701 R D_S01764691 R D_S01764680S S R D_S01764668 R D_S01764642 R D_S01764568 R D_S01764554 RD_S01764546 R D_S01764543 R D_S01764533 R D_S01764505 H N V RD_S01764502 A R D_S01764500 R D_S01764196 S R D_S01763985 R D_S01632237R D_S01632121 A R D_S01631557 R D_S01614407 R D_S01613034 R RD_S01602925 R D_S01601459 R D_S01599948 R D_S01532970 R R D_S0153204 H NS R D_S01467003 R D_S01466770 R D_S01466712 R D_S01466681 H D_S014657862I R D_S01457892 R D_S01443366 R D_S01442158 R L D_S01442132 RD_S-1764685 R BY2_Cry2Ab R Sample 64 70 76 79 80 89 93 96 97 100 101 107111 118 119 122 123 124 125 126 130 135 137 139 Cry2Ab* K V S R N N D RE K F N L L T Q A N V E R F N N Cry2Ab wt seq D_S02838648 S L ED_S02838640 S E P D_S02838632 P D_S02838470 Q D_S02838434 S LD_S02838328 D_S02838327 S E D_S02838310 D_S01766073 D_S01766026 W ED_S01765621 G D D_S01765509 D_S01765496 I D_s01765255 Q D_S01765119 QD_S01765112 D D_S01765104 H W D_S01765063 Q D D_S01765008 W GD_S01764977 W Q D_S01764947 K S Q D_S01764787 W Y D_S01764723 W G MD_S01764701 Q D D_S01764691 D_S01764680 D_S01764668 W G R D_S01764642 LQ D_S01764568 G I D_S01764554 S R D_S01764546 Q D_S01764543 E SD_S01764533 W T D_S01764505 S D_S01764502 W G Q R D_S01764500 G TD_S01764196 S G D_S01763985 D_S01632237 S D_S01632121 S D_S01631557 S VH D_S01614407 G R D_S01613034 D_S01602925 S D_S01601459 S E D_S01599948S G D_S01532970 D_S0153204 G D_S01467003 D_S01466770 D_S01466712D_S01466681 D_S014657862 S R H D_S01457892 D_S01443366 D_S01442158D_S01442132 D_S-1764685 BY2_Cry2Ab S E Sample 140 141 142 144 153 154160 162 164 166 167 168 169 172 178 184 187 191 192 193 197 201 Cry2Ab*R N A P T M N L Q Q M Q G L F L S D V I D I Cry2Ab wt seq D_S02838648 GD_S02838640 D I D_S02838632 L S I Q D_S02838470 T D_S02838434 LD_S02838328 M D_S02838327 L D T D_S02838310 D_S01766073 D_S01766026 Q MD_S01765621 V V D_S01765509 R V V D_S01765496 D_s01765255 V M VD_S01765119 L D_S01765112 A D_S01765104 S M E D_S01765063 D_S01765008 SV M V D_S01764977 D_S01764947 D_S01764787 V D_S01764723 Q L R V VD_S01764701 D_S01764691 D_S01764680 R M V D_S01764668 M D_S01764642 V MV D_S01764568 D_S01764554 A D_S01764546 D_S01764543 D_S01764533 VD_S01764505 D_S01764502 Q G V D_S01764500 Q V D_S01764196 R SD_S01763985 D_S01632237 R Q D_S01632121 D_S01631557 D_S01614407D_S01613034 D_S01602925 D_S01601459 D_S01599948 D_S01532970 D_S0153204D_S01467003 D_S01466770 D_S01466712 T D_S01466681 D_S014657862 MD_S01457892 D_S01443366 D_S01442158 D_S01442132 D_S-1764685 BY2_Cry2AbSample 205 210 211 212 214 215 216 218 219 221 226 229 230 233 234 237238 240 241 242 244 273 Cry2Ab* T R D Y K N Y R D S N Q S K G T R H D TE G Cry2Ab wt seq M D_S02838648 T D_S02838640 T T D_S02838632 TD_S02838470 T D_S02838434 R T D_S02838328 T D_S02838327 T D_S02838310 QT T D_S01766073 D_S01766026 T D_S01765621 T T D_S01765509 T D_S01765496T D_s01765255 T D_S01765119 T D_S01765112 Q N H R T R D_S01765104 TD_S01765063 G T D_S01765008 T D_S01764977 T K D_S01764947 T D_S01764787A T D_S01764723 T S D_S01764701 G T D_S01764691 T D_S01764680 R TD_S01764668 E T D_S01764642 E T D_S01764568 T D_S01764554 Q S TD_S01764546 A T D_S01764543 T T D_S01764533 T D_S01764505 T D_S01764502A G T D_S01764500 A T D_S01764196 Q R G T D_S01763985 H T T D_S01632237N T T D_S01632121 Q T T D_S01631557 R T D_S01614407 T T D_S01613034 N RT T D_S01602925 R T T D_S01601459 Q N T T D_S01599948 T T D_S01532970 FD_S0153204 Q N H R T R T D_S01467003 T D_S01466770 T D_S01466712 TD_S01466681 T D_S014657862 E T D_S01457892 T D_S01443366 T D_S01442158 TD_S01442132 T D_S-1764685 W BY2_Cry2Ab N R T T Sample 278 279 283 284285 286 288 291 298 302 305 307 310 311 317 319 321 323 325 330 331 344Cry2Ab* A S P Q Q T S S Y Q S Y N G L N F N V S T S Cry2Ab wt seqD_S02838648 D_S02838640 G D_S02838632 D_S02838470 H D_S02838434D_S02838328 G D_S02838327 D_S02838310 D_S01766073 A D_S01766026D_S01765621 G D_S01765509 G D_S01765496 L D_s01765255 G D_S01765119D_S01765112 Q L D_S01765104 G D_S01765063 G D_S01765008 G D_S01764977D_S01764947 D_S01764787 G D_S01764723 S D_S01764701 G D_S01764691D_S01764680 H G D_S01764668 G P D_S01764642 S D_S01764568 D_S01764554 AD_S01764546 D_S01764543 C D_S01764533 G D_S01764505 Q G D_S01764502 SD_S01764500 T A D_S01764196 D_S01763985 V T N D_S01632237 S D_S01632121H D_S01631557 D_S01614407 S D_S01613034 V T N D_S01602925 D_S01601459 RN D_S01599948 D_S01532970 T D_S0153204 Q T D_S01467003 D_S01466770D_S01466712 R D_S01466681 D_S014657862 D D_S01457892 D_S01443366D_S01442158 D_S01442132 D_S-1764685 R R BY2_Cry2Ab Sample 347 358 362367 383 386 389 391 399 401 403 405 407 408 413 420 435 436 444 445 447448 Cry2Ab* I N N L S E A V F T L L S G R P R N H Y E I Cry2Ab wt seqD_S02838648 D_S02838640 D_S02838632 D_S02838470 D_S02838434 D_S02838328D_S02838327 D_S02838310 D_S01766073 T D_S01766026 D_S01765621D_S01765509 D_S01765496 D_s01765255 V D_S01765119 D_S01765112 G ND_S01765104 S I I R D_S01765063 D_S01765008 S I I C D_S01764977D_S01764947 D_S01764787 D_S01764723 K D_S01764701 D_S01764691D_S01764680 D_S01764668 S I I D_S01764642 D_S01764568 D_S01764554D_S01764546 T D_S01764543 D_S01764533 D_S01764505 Y F D_S01764502D_S01764500 D_S01764196 D_S01763985 D_S01632237 D_S01632121 D_S01631557D_S01614407 D_S01613034 D_S01602925 D_S01601459 D_S01599948 KD_S01532970 V S L Q D_S0153204 G N D_S01467003 S D D_S01466770 DD_S01466712 D_S01466681 D_S014657862 L D_S01457892 D_S01443366D_S01442158 D_S01442132 D_S-1764685 S T R BY2_Cry2Ab Sample 459 461 476490 491 492 497 498 500 508 513 517 529 530 537 538 543 545 553 560 566Cry2Ab* G A H N D Y I S I Q I F N N L R S N I V V Cry2Ab wt seqD_S02838648 K D_S02838640 V D_S02838632 D_S02838470 D_S02838434D_S02838328 D_S02838327 D_S02838310 G D_S01766073 D_S01766026D_S01765621 V D_S01765509 D_S01765496 D_s01765255 D_S01765119D_S01765112 L Y E S D_S01765104 G — D_S01765063 D_S01765008 GD_S01764977 P D_S01764947 D_S01764787 A D_S01764723 D_S01764701D_S01764691 D_S01764680 D_S01764668 D_S01764642 W D_S01764568D_S01764554 D_S01764546 D_S01764543 S D_S01764533 D_S01764505D_S01764502 D_S01764500 T D_S01764196 A D_S01763985 D_S01632237D_S01632121 D_S01631557 D_S01614407 R D_S01613034 D_S01602925D_S01601459 D_S01599948 D D_S01532970 L E D_S0153204 P D A D_S01467003 FL D_S01466770 L T F L D_S01466712 T L D_S01466681 D_S014657862D_S01457892 G F D_S01443366 M T L D_S01442158 A F D_S01442132 TD_S-1764685 H Y BY2_Cry2Ab Sample 567 568 569 582 583 591 592 593 595598 600 603 612 619 623 624 625 630 631 633 Cry2Ab* Y T A N D I N I N AS D L D I M L I S L Cry2Ab wt seq D_S02838648 D_S02838640 D_S02838632D_S02838470 I D_S02838434 D_S02838328 H F I D_S02838327 D_S02838310 AD_S01766073 D_S01766026 D F D_S01765621 I D_S01765509 A F I D_S01765496M I D_s01765255 M I D_S01765119 D_S01765112 D_S01765104 M D_S01765063 FT D_S01765008 M F I D_S01764977 M F I D_S01764947 M V I D_S01764787 M FD_S01764723 D_S01764701 F T D_S01764691 D_S01764680 M I D_S01764668 MD_S01764642 K M F I D_S01764568 V M V I D_S01764554 D_S01764546 MD_S01764543 D_S01764533 I D_S01764505 D_S01764502 D_S01764500 F ID_S01764196 D_S01763985 D_S01632237 D_S01632121 D_S01631557 D_S01614407D_S01613034 D_S01602925 D_S01601459 D_S01599948 S D_S01532970 F L PD_S0153204 Y F D_S01467003 G D_S01466770 N I D_S01466712 T F D_S01466681E D_S014657862 T D_S01457892 I D_S01443366 V M F G D_S01442158D_S01442132 V M F I D_S-1764685 N BY2_Cry2Ab

TABLE 6 Amino acid sequence changes of clones of interest isolated fromDNA shuffling using clone 44 as parent Sample 64 143 149 189 192 216 217225 228 290 345 358 383 418 527 553 600 603 616 619 631 D_S00503970 K VS I V Y T I Y T G N S Y E I S D T D P (Parental Clone 44) D_S01765068 DN E S D_S01573294 H S S D_S01572908 A D_S01572905 P A D_S01561856 L H AD_S01561489 A F G D_S01561330 R H D_S01765221 R D G D_S01561293 R V M

TABLE 7 Sequence of clones of interest isolated from DNA shuffling usingclone 473R as parent Sample 4 12 17 29 31 32 34 38 51 93 96 108 124 137139 140 141 142 143 144 147 153 167 D_S01037677 N T Y H S L T E V D R TN N N R N A V P I T M (parental clone 473) D_S01765231 D_S01764643 PD_S01572374 D_S01571875 D_S01766010 H P T I S H D_S01572168 P T Q T T RL D_S01765242 G L D_S01764953 H R G S V L D_S01764728 I A M LD_S01764758 L D_S01571529 S A V D_S01571315 C V D_S01570809 DD_S01570568 D_S01570511 A Sample 168 169 170 177 189 195 223 226 250 266296 306 315 323 324 350 355 356 357 358 362 372 378 D_S01037677 Q G Y LI N Y N F Q F N A N I G S P F N S T W (parental clone 473) D_S01765231 SD_S01764643 D_S01572374 S S D_S01571875 S D_S01766010 V H S S RD_S01572168 H R D_S01765242 N N R D_S01764953 R H D_S01764728 R D P VD_S01764758 G E S V R T A G N S D_S01571529 E R D D D_S01571315 G FD_S01570809 D_S01570568 V R D_S01570511 Sample 384 390 425 434 446 447451 455 474 479 492 504 505 527 553 555 565 570 599 611 616 623D_S01037677 D T R V N Q I S N H Y Q V E I N R T S T T I (parental clone473) D_S01765231 R D_S01764643 G D_S01572374 D_S01571875 Q H RD_S01766010 P R A D_S01572168 A D_S01765242 G D_S01764953 A D_S01764728D A V D_S01764758 A I D_S01571529 R Q D_S01571315 T D_S01570809 SD_S01570568 H V D_S01570511 S

1. An isolated polypeptide having insecticidal activity selected fromthe group consisting of: a) a polypeptide comprising the amino acidsequence of SEQ ID NO: 22; and b) a polypeptide encoded by a nucleicacid molecule comprising the nucleotide sequence set forth in SEQ ID NO:21.
 2. The isolated polypeptide of claim 1, wherein the polypeptide hasinsecticidal activity against Diptera or Lepidoptera insects.
 3. Acomposition comprising the isolated and purified polypeptide of SEQ IDNO: 22, and an agent, wherein the agent is selected from the groupconsisting of spreader-sticker adjuvants, stabilizing agents,insecticidal agents, diluents, surfactants, emulsifiers, anddispersants; said polypeptide having insecticidal activity.
 4. Thecomposition of claim 3, wherein the composition further comprisesisolated cells.
 5. The composition of claim 4, wherein the cells arewhole or lysed.
 6. The composition of claim 4, wherein the cells are insuspension or are pelleted.
 7. The composition of claim 3, wherein thecomposition further comprises a spore-crystal complex of Bacillusthuringiensis.